Project Summary/Abstract Bi-directional vesicular transport is vital in all eukaryotic cells to deliver proteins to secretory organelles and the cell surface, and for release from the cell. It is estimated that 30% of the mammalian proteomes must traffic the secretory pathway. GBF1 (Golgi localized Brefeldin A-sensitive Factor1) is a key regulator of retrograde traffic from the Golgi to the ER, and GBF1 activity is required for the establishment and maintenance of the secretory pathway. GBF1 belongs to a family of large Guanine nucleotide Exchange Factors (GEFs) and is an enzyme that facilitates GDP/GTP exchange on the ARF subfamily of small Ras-like GTPases. GBF1-mediated ARF activation is required for the formation of retrograde COPI vesicles, and GBF1 represents an upstream regulator of COPI vesicle formation as it dictates the time and site of vesicle formation by restricting ARF activation. Yet, despite the critical importance of GBF1 in cellular homeostasis, we remain ignorant of how GBF1 itself is regulated in cells. Specifically, we do not know how cells signal to GBF1 to ?notify? it of a cellular need for retrograde traffic and what mechanisms ensure that GBF1 initiates COPI vesicle formation only at the right time and the right place. This proposal aims to illuminate this enigma. We will test the hypothesis that cells have mechanisms to inhibit GBF1 activity to prevent spurious ARF activation, but release such inhibition in a time and site-restricted manner in response to a signaling pathway that indicates retrograde traffic demand. Our goal is to define how cells translate their need for ARF activation and membrane transport into space- and time-restricted GBF1 function. We propose 3 specific aims to identify the processes and signaling pathways that regulate GBF1 function in an essential traffic routing in all cells, retrograde COPI traffic that is vital for the homeostasis of the secretory pathway. In Aim 1, we will identify the mechanisms that selectively target GBF1 to Golgi membranes by identifying the intrinsic targeting information within GBF1 and the membrane components that mark membrane sites for GBF1 recruitment. In Aim 2, we will define the mechanisms that regulate GBF1 catalytic activity at the membrane by assessing the role of phosphatidylinositol phosphates (PIPs) in regulating GBF1 catalytic activity. In Aim 3, we will determine the signaling pathways that coordinate GBF1 function with the need for COPI traffic by defining the role of KDEL-R activation and the PKA and SFK pathways on GBF1 membrane association and catalytic activity. GBF1 is ubiquitously expressed and critically important to cell and organismal health; GBF1 depletion from cultured cells causes death and a mouse or Drosophila knockout is embryonic lethal. GBF1 is also important in pathological contexts since it is essential for migration of glioblastoma cells and for replication of human pathogenic enteroviruses. Our studies will provide critical new knowledge of GBF1 regulation in basic cellular physiology and will inform strategies for the design of therapeutic intervention to control GBF1-mediated events in pathological contexts.